Fluorescence Spectroscopy Flashcards
What do we mean by fluorescence?
The property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of a LONGER WAVELENGTH after a brief interval.
What are the three events in the fluorescence process?
*Excitation of a molecule by an incoming photon (10^-15 sec) (S0 -> S1 or S2)
*vibrational relaxation (or internal conversion) of excited state electrons to lowest vibrational level of an excited state (10^-12 sec) (relaxed to lowest vibrational sub level of first excited state (S1)
*emission of longer wavelength photon and return of molecule to ground state (10^-9 sec)- return from lowest vibrational level in S1 to any vibrational level in ground state
What is ground state denoted by?
S0
What are the excited energy levels?
S1 and S2 (any vibrational sub level in these states)
What is meant by vibrational relaxation?
Excited electron transitions from higher vibrational sub level to lower vibrational sub level within same electronic state
What is meant by internal conversion?
Excited electron transitions from vibrational level in one electronic state to another vibrational level in lower electronic state I.e S2>S1
What is meant by inter system crossing?
Transition between electronic states of different spin multiplicity eg from excited singlet state to an excited triplet state
What is meant by phosphorescence?
Radiative transition from an excited triplet state to a singlet ground state
What is the mirror image rule?
Vibrational energy level spacing is similar for ground and excited states. This means that absorption and emission spectrums mirror each other (only S1)
What is an exemption to mirror image rule?
If there is excitation by high energy photons which leads to excitation to higher electronic and vibrational levels (S2 and S3)- fluorophore quickly loses excess energy as it relaxes to lowest vibrational level of first excited state.
What are characteristics of an ideal fluorescent molecule?
*high extinction coefficient e
*high fluorescence quantum yield- ratio between photons emitter and absorbed
*large Stokes shift- difference between peak excitation and peak emission wavelengths
*long wavelength of emission
§NB- usually contains multiple conjugated (-C=C-) and/or rigid structure but hard to predict from chemical structure I.e noradrenaline exhibits 20x more intense fluorescence than adrenaline but same number of -C=C-
What factors interfere with fluorescence intensity?
*quenching- interaction of excited state fluorophore with surrounding eg energy transfer
*concentration- at high solution conc intensity of excitation light NOT UNIFORM through sample. Also some light emitted light can be reabsorbed by other uneducated molecules in solution- non-linearity
*temperature- increased frequency of collisions between fluorophores and solvent molecules at high temp increases probability of deactivation.
*pH- some molecules may be fluorescent in acidic media but not basic eg phenols
Others- turbidity, heavy atoms in solution, bubbles
What are applications of fluorescence spectroscopy?
*Quantitive analysis- fluoro intensity proportional to concentration of fluorophore (to a limit)- can quantify amount of something in a drug or solution.
*use fluorophores to analyse non-fluorescent drugs- mix together and quantify fluorophore intensity as usual.
*fluorescence imaging- use to make cells stand out under microscope
What are advantages of fluorescence spectroscopy?
*High sensitivity
*high specificity- determine fluorescent drugs in non fluorescent excipients
What are limitations of fluorescence spectroscopy?
*limited to fluorescent molecules only
*temperature dependent
*interference by high conc solutions- self quenching effects
